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Source 2: Scientists Identify Point of Entry for Deadly Ebola Virus

September 30, 2012 Leave a comment

Ebola virus, the cause of Ebola hemorrhagic fever (EHF), is one of the deadliest known viruses affecting humans. Like anthrax and smallpox virus, Ebola virus is classified by the U.S. Centers for Disease Control and Prevention (CDC) as a category A bioterrorism agent. Currently, there is no vaccine to prevent EHF, and patients are treated only for their symptoms.Although outbreaks are rare, Ebola virus, the cause of Ebola hemorrhagic fever (EHF), is one of the deadliest known viruses affecting humans. According to the World Health Organization (WHO), approximately 1,850 EHF cases with more than 1,200 deaths have been documented since the virus was identified in 1976.

This negatively-stained transmission electron micrograph (TEM) revealed some of the ultrastructural curvilinear morphologic features displayed by the Ebola virus discovered from the Ivory Coast of Africa. (Credit: Charles Humphrey). (up)

EHF’s clinical presentation can be devastating: fever, intense weakness, and joint and muscle aches progress to diarrhea, vomiting, and in some cases, internal and external bleeding caused by disintegrating blood vessels. Currently, there is no approved vaccine and patients are treated only for their symptoms. Like anthrax and smallpox virus, Ebola virus is classified as a category A bioterrorism agent by the U.S. Centers for Disease Control and Prevention (CDC).

Until now, however, researchers had only a limited understanding of how Ebola virus gains entry to a host cell.

Using an unusual human cell line, Whitehead Institute scientists and collaborators from Harvard Medical School, Albert Einstein College of Medicine and U.S. Army Medical Research Institute of Infectious Diseases, have identified the Niemann-Pick C1 (NPC1) protein as crucial for Ebola virus to enter cells and begin replicating. The discovery may offer a new and better approach for the development of antiviral therapeutics, as it would target a structure in the host cell rather than a viral component.

The findings are reported online in Nature this week.

Where all of us inherit one copy of each chromosome from each of our two parents, cell lines exist with only a single set, and thus with a single copy of each individual gene, instead of the usual two. Using an unusual human cell line of this type, Whitehead Institute researchers and their collaborators performed a genetic screen and identified a protein used by Ebola virus to gain entry into cells and begin replicating. The discovery may offer a new approach for the development of antiviral therapeutics.

“Right now, people make therapeutics to inactivate the pathogen itself. But the problem is that pathogens can quickly change and escape detection and elimination by the immune system,” says former Whitehead Fellow Thijn Brummelkamp, now a group leader at the Netherlands Cancer Institute (NKI). “Here we get a good idea of the host genes that are needed for the pathogen to enter the cell for replication. Perhaps by generating therapeutics against those host factors, we would have a more stable target for antiviral drugs.”

The method developed by the Brummelkamp lab to identify host factors relies on gene disruption — knocking out gene function in the host cells, one gene at a time — and documenting which cells survive due to mutations that afford protection from viral entry.

But human cells are diploid with two copies of each chromosome and its genes. Researchers can reliably target and knock out one copy of a gene, but doing so for both copies is far more difficult and time-consuming. If only a single copy is silenced, the other continues to function normally and masks any effect of the knockout.

To sidestep this obstacle, Jan Carette, a first co-author on the Nature paper and a former postdoctoral researcher in the Brummelkamp lab, employed a technique he had previously applied to study the cytolethal distending toxin (CDT) family that is secreted by multiple pathogenic bacteria, including Escherichia coli, Shigella dysenteriae, and Haemophilus ducreyi. Each bacterial species has developed its own twists on the CDT structure, which may link to the target tissues of the toxin’s bacterium.

In his CDT work published in Nature Biotechnology, Carette together with co-lead authors of Whitehead Member Hidde Ploegh’s lab, used a line of haploid cells isolated from a chronic myeloid leukemia (CML) patient. Because these cells, called KBM7 cells, have only one copy of each chromosome except chromosome 8, the researchers could disrupt the expression of each gene and screen for mutants with the desired properties, in this case survival of a lethal dose of toxin.

After knocking out individual genes by disrupting the normal structure of the gene, the resulting mutant KBM7 cells were exposed to various CDTs. In the cells that survived, Carette and coauthors knew that genes that had been disrupted were somehow crucial to CDT intoxication. By analyzing the surviving cell’s genomes, Carette and coauthors identified ten human proteins that are used by CDTs during intoxication, and those host factors seem to be tailored to each CDT’s targeted cell.

“I found it surprising that there is quite some specificity in the entry routes for each toxin,” says Carette. “If you take CDTs that are very similar to each other in structure, you could still see significant differences in the host factors they require to do their job. So it seems that every pathogen evolved a specific and unique way of its toxin entering the cells.”

To study Ebola virus, Carette and co-lead authors from Harvard Medical School and the Albert Einstein College of Medicine made use of an otherwise harmless virus cloaked in the Ebola virus glycoprotein coat. Using this virus and by altering the haploid cells somewhat, Carette and coauthors were able to pinpoint the cellular genes that Ebola virus relies on to enter the cell.

Carette and coauthors identified as necessary for Ebola virus entry several genes involved in organelles that transport and recycle proteins. One gene in particular stood out, NPC1, which codes for a cholesterol transport protein, and is necessary for the virus to enter the cell’s cytoplasm for replication. Mutations in this gene cause a form of Niemann-Pick disease, an ultimately fatal neurological disorder diagnosed mainly in children.

Collaborators at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) tested the effects of active Ebola virus on mice that had one copy of the NPC1 gene knocked out. Control mice, with two functioning copies of the NPC1 gene, quickly succumbed to infection, while the NPC1 knockout mice were largely protected from the virus.

“This is pretty unexpected,” says Carette, who is currently an Acting Assistant Professor in Microbiology & Immunology at Stanford School of Medicine. “This might imply that genetic mutations in the NPC1 gene in humans could make some people resistant to this very deadly virus. And now that we know that NPC1 is an Ebola virus host factor, it provides a strong platform from which to start developing new antivirals.”

This research was supported by the National Institutes of Health (NIH), the U.S. Army, Boehringer Ingelheim Fonds and a Burroughs Wellcome Award.

Story Source:

The above story is reprinted from materials provided by Whitehead Institute for Biomedical Research, via ScienceDaily. The original article was written by Nicole Giese.

Journal References:

  • Jan E. Carette, Matthijs Raaben, Anthony C. Wong, Andrew S. Herbert, Gregor Obernosterer, Nirupama Mulherkar, Ana I. Kuehne, Philip J. Kranzusch, April M. Griffin, Gordon Ruthel, Paola Dal Cin, John M. Dye, Sean P. Whelan, Kartik Chandran, Thijn R. Brummelkamp. Ebola virus entry requires the cholesterol transporter Niemann–Pick C1. Nature, 2011; DOI: 10.1038/nature10348
  • Jan E Carette, Carla P Guimaraes, Irene Wuethrich, Vincent A Blomen, Malini Varadarajan, Chong Sun, George Bell, Bingbing Yuan, Markus K Muellner, Sebastian M Nijman, Hidde L Ploegh, Thijn R Brummelkamp. Global gene disruption in human cells to assign genes to phenotypes by deep sequencing. Nature Biotechnology, 2011; 29 (6): 542 DOI: 10.1038/nbt.1857
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Source 1: Researchers Find ‘Key’ Used by Ebola Virus to Unlock Cells and Spread Deadly Infection

September 30, 2012 Leave a comment

Researchers at Albert Einstein College of Medicine of Yeshiva University have helped identify a cellular protein that is critical for infection by the deadly Ebola virus. The findings, published in the August 24 online edition of Nature, suggest a possible strategy for blocking infection due to Ebola virus, one of the world’s most lethal viruses and a potential bioterrorism agent.

The study was a collaborative effort involving scientists from Einstein, the Whitehead Institute for Biomedical Research, Harvard Medical School, and the U.S. Army Medical Research Institute of Infectious Diseases

Ebola virus is notorious for killing up to 90 percent of the people it infects. Ebola hemorrhagic fever — the severe, usually fatal disease that Ebola virus causes in humans and in nonhuman primates — first emerged in 1976 in villages along the Ebola River in the Sudan and the Democratic Republic of the Congo, Africa. Since then, about two dozen outbreaks have occurred.

This drawing illustrates the sequence of events from the time the Ebola virus first enters the host cell (top) until the virus gains its release into the cytoplasm, where it can multiply (bottom). Researchers have shown that Ebola exists in the lysosome and enters the cytoplasm by interacting with NPC1 protein molecules (orange) embedded in the lysosomal membrane. (Credit: Image courtesy of Albert Einstein College of Medicine) (right)

Though Ebola and Marburg hemorrhagic fevers are fortunately rare diseases, “even small outbreaks of Ebola or Marburg virus can cause fear and panic,” said co-senior author Kartik Chandran, Ph.D., assistant professor of microbiology & immunology at Einstein “And then there’s the worry that these viruses could be used for bioterrorism.”

Ebola virus’s ability to enter cells is reminiscent of the Trojan Horse used by the ancient Greeks to defeat their archenemies. Ebola virus binds to the host cell’s outer membrane, and a portion of host cell membrane then surrounds the virus and pinches off, creating an endosome — a membrane-bound bubble inside the cell (see image). Endosomes carry their viral stowaways deep within the cell and eventually mature into lysosomes — tiny enzyme-filled structures that digest and recycle cellular debris.

The viruses captive in the lysosome manage to escape destruction by exploiting components of the cell to gain entry to the cytoplasm, the substance between the cell membrane and the nucleus where the virus can replicate. But the identities of many of these components have remained unknown.

In seeking the answer, Einstein researchers and colleagues searched for proteins that Ebola virus might exploit to enter the cell’s cytoplasm. One such cellular protein, known as Niemann-Pick C1 (NPC1), stood out.

“We found that if your cells don’t make this protein, they cannot be infected by Ebola virus,” said Dr. Chandran. “Obviously it’s very early days, but we think our discovery has created a real therapeutic opportunity.” At present, there are no drugs available to treat people who have been infected with Ebola virus or approved vaccines to prevent illness.”

The NPC1 protein is embedded within cell membranes, where it helps transport cholesterol within the cell. However, the absence of NPC1 due to gene mutations causes a rare degenerative disorder called Niemann-Pick disease, in which cells become clogged up with cholesterol and eventually die.

To confirm their finding that NPC1 is crucial for Ebola virus infection, the researchers challenged mice carrying a mutation in NPC1 with Ebola virus. Remarkably, most of these mutant mice survived the challenge with this normally deadly virus. Similarly, fibroblast cells (found in connective tissue) from people with Niemann-Pick disease were resistant to Ebola virus infection, as were human cells from other organs that were manipulated to reduce the amount of NPC1 they contained.

The researchers also tested whether other major viruses need NPC1 to infect human cells. Only Ebola virus and its close relative, Marburg virus, were found to require the presence of NPC1 protein for infection. Like Ebola virus, Marburg virus also needs NPC1 to kill mice.

“Our work suggests that these viruses need NPC1, which is embedded in the lysosomal membrane, to escape from the lysosome into the cytoplasm,” said Dr. Chandran. “We are now testing that hypothesis in the laboratory.”

The discovery of NPC1’s crucial role in Ebola infection raises the possibility that Ebola and Marburg virus outbreaks could be thwarted by a drug that blocks the action of NPC1. “Even though such a treatment would also block the cholesterol transport pathway, we think it would be tolerable,” said Dr. Chandran. “Most outbreaks are short-lived, so treatment would be needed for only a short time.” Einstein, in conjunction with the Whitehead Institute of Biomedical Research and Harvard Medical School, has filed a patent application related to this research that is available for licensing to partners interested in further developing and commercializing this technology.

Remarkably, an anti-Ebola virus inhibitor Dr. Chandran found as a postdoctoral fellow at the Brigham and Women’s Hospital in Boston, MA turns out to be just such an NPC1 blocker, as described in a separate manuscript by Côté and co-workers to be published in the same issue of Nature.

Story Source:

The above story is reprinted from materials provided by Albert Einstein College of Medicine, via ScienceDaily.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

Journal Reference:

Jan E. Carette, Matthijs Raaben, Anthony C. Wong, Andrew S. Herbert, Gregor Obernosterer, Nirupama Mulherkar, Ana I. Kuehne, Philip J. Kranzusch, April M. Griffin, Gordon Ruthel, Paola Dal Cin, John M. Dye, Sean P. Whelan, Kartik Chandran, Thijn R. Brummelkamp. Ebola virus entry requires the cholesterol transporter Niemann–Pick C1. Nature, 2011; DOI: 10.1038/nature10348

Novartis Cancer Drugs Fight Deadly Ebola Virus in Lab, Researchers Find


Two Novartis AG leukemia drugs, Gleevec and Tasigna, fought the deadly Ebola virus in laboratory experiments, suggesting the products could be used against a disease for which there are no treatments.

The two medicines stopped the release of viral particles from infected cells in lab dishes, a step that in a person may prevent Ebola from spreading in the body and give the immune system time to control it, researchers from the U.S. National Institute of Allergy and Infectious Diseases wrote in the journal Science Translational Medicine today.

There’s no cure and no vaccine for Ebola, a virus that causes high fever, diarrhea, vomiting and internal and external bleeding. Death can ensue within days, and outbreaks in Africa have recorded fatality rates of as much as 90 percent, according to the World Health Organization.

In some forms of leukemia, Gleevec and Tasigna reduce levels of a protein called Bcr-Abl that causes malignant white blood cells to multiply.

The researchers found that Ebola uses a related protein called c-Abl1 tyrosine kinase to regulate its own reproduction. They showed that by blocking c-Abl1, Tasigna may reduce the pathogen’s ability to replicate by as much as 10,000-fold. In addition to showing how the two drugs might be used to treat infected patients, the findings also suggest that new medicines could be developed to target c-Abl1, they wrote.

Gleevec and Tasigna, also known as imatinib and nilotinib, earned Basel, Switzerland-based Novartis a combined $5.45 billion in sales last year. Gleevec is sold as Glivec outside the U.S.

Source: Yahoo! Health

Cancer Drugs Could Halt Ebola Virus


Some cancer drugs used to treat patients with leukemia may also help stop the Ebola virus and give the body time to control the infection before it turns deadly, US researchers said on Wednesday.

The much-feared Ebola virus emerged in Africa in the 1970s and can incite a hemorrhagic fever which causes a person to bleed to death in up to 90 percent of cases.

While rare, the Ebola virus is considered a potential weapon for bioterrorists because it is so highly contagious, so lethal and has no standard treatment.

But a pair of well-known drugs that have been used to treat leukemia — known as nilotinib and imatinib — appear to have some success in stopping the virus from replicating in human cells.

Lead researcher Mayra Garcia of the US National Institute of Allergy and Infectious Diseases and colleagues reported their finding in Wednesday’s edition of the journal Science Translational Medicine.

By experimenting with human embryonic kidney cells in a lab, they found that a protein called c-Abl1 tyrosine kinase was a key regulator in whether the Ebola virus could replicate or not.

The leukemia drugs work by stopping that protein’s activity. In turn, a viral protein called VP40 stopped the release of viral particles from the infected cells, a process known as filovirus budding.

“Drugs that target filovirus budding would be expected to reduce the spread of infection, giving the immune system time to control the infection,” the study authors wrote.

“Our results suggest that short-term administration of nilotinib or imatinib may be useful in treating Ebola virus infections.”

Imatinib, which is marketed as Gleevec and Glivec, is used to treat chronic myelogenous leukemia in humans, a disease which is caused by dysregulation of c-Abl enzyme.

Nilotinib, also known as Tasigna, has been used in chronic myelogenous leukemia patients who are resistant to imatinib.

Both “have reasonable safety profiles, although some cardiac toxicity has been reported with long-term administration in a small number of patients,” the study added.

According to the UN’s World Health Organization (WHO), about 1,850 cases of Ebola, with some 1,200 deaths, have occurred since 1976.

The virus has a natural reservoir in several species of African fruit bat. Gorillas and other non-human primates are also susceptible to the disease.

Source: Bloomberg